Activation of Ras is a key link between extracellular stimuli and downstream events that lead to cell proliferation and differentiation. Mutations leading to inappropriate Ras activation are known to cause leukemia and solid tumors, as well as developmental disorders such as Noonan Syndrome. Ras cycles between active GTP-bound and inactive GDP-bound states, which is controlled by Ras guanine nucleotide exchange factors (GEFs) and guanine nucleotide activating proteins (GAPs) and insensitivity of Ras to these proteins, or inappropriate activation of GEFs or GAPS often underlie these diseases. T-cell receptor activation can cause varying degrees of Ras stimulation through the combined action of two GEFs, RasGrp1 and SOS, which leads to cell proliferation or death, depending on the strength and duration of the extracellular signal. This process of positive or negative selection of immature T cells (thymocytes) is a key pathway for adaptive immunity and the prevention of autoimmune responses. The long-term goal of this project is to reconstitute the T-cell receptor signaling pathway in vitro on model membranes to understand the structural and biochemical basis for positive and negative thymocyte selection. For Ras activation to occur, RasGrp1 and/or SOS must first be activated by upstream signals, but the molecular basis for many aspects of RasGEF activation have not been determined. It is also unclear why two different RasGEFs are necessary in this signaling cascade. This proposal will investigate the hypothesis that SOS and RasGrp1 are activated by unique signals that allow for cooperative Ras activation. The following specific aims will test this hypothesis. 1) The impact of SOS membrane recruitment by activated receptor complexes on catalytic activity will be elucidated using a fluorescence nucleotide exchange assay with membrane anchored Ras. 2) Regulatory mechanisms of RasGrp1 will be elucidated by measuring nucleotide exchange activity of different RasGrp1 constructs that contain only the catalytic domain or this domain with the C-terminal EF-hands, C1 domain and coiled-coil motif. In addition, the presence of auxiliary Ras binding sites will be investigated to determine if RasGrp1 contains an allosteric binding pocket responsible for a positive feedback loop such as that found in SOS. Finally, the ability of RasGrp1 and SOS to cooperatively activate Ras will be tested by combining the two proteins in nucleotide exchange experiments. 3) The structural basis for RasGrp1 catalysis and flanking domain effects will be probed using X-ray crystallography. These comparative studies between RasGrp1 and SOS will provide a structural and biochemical basis for how Ras activation occurs in T-cells, and how activation of two enzymes with similar biochemical activities can differentially induce distinct cell fates. PUBLIC HEALTH RELEVANCE: Ras is a key protein switch that is activated in signaling cascades important for cell growth, development and death. Activating Ras mutations are found in ~30% of cancers and improper Ras regulation can also result in developmental disorders including Noonan Syndrome. Because of the importance of Ras activity in human disease, it is important to understand the molecular mechanisms responsible for precisely tuning its functions at the membrane.